There is growing evidence that lack of proper N-glycosylation can induce serious medical problems. It is also known that secretory glycoproteins that do not fold correctly can produce metabolic disorders. The objective of this program, using yeast as a model for higher eukaryotes, is to study two yeast enzymes involved in the metabolic fate of glycoproteins. One is protein disulfide isomerase (PDI), a lumenal enzyme of the endoplasmic reticulum (ER) that mediates correct disulfide bond formation in newly synthesized glycoproteins (and proteins). The other is peptide N-glycanase (PNGase), a cytoplasmic enzyme that removes oligosaccharide chains from glycopeptides and possibly unfolded glycoproteins. A collaborative effort will be made to obtain the three dimensional structure of PDI. Two major objectives are to understand how this enzyme acts as a sulfhydryl oxidase and as a disulfide isomerase, and if it acts as a chaperone. With respect to PNGase, it has been observed that it interacts with the proteosome via only one protein, Rad 23p. Therefore, in a collaborative effort, attempts will be made to crystallize and obtain the three-dimensional structures of PNGase and of Rad23p. Knowledge of these structures will be very important because deletion analyses have established which domains of these proteins are essential for their interaction. PNGase in mice and humans contains a N-terminal extension that is required for interaction with the mouse HR23Bp, and interestingly, with a number of other proteins related to the proteosome. To understand these multiple protein interactions, three approaches will be taken. One is to isolate the proteosome and determine if mHR23Bp, mPNGase and other proteins involved in ubiquitination are in this proteosome complex. The second approach will be to determine the sequence of reactions that result in the destruction of misfolded glycoproteins by a reconstitution approach involving the individual components. The third will be deletion analysis of these various mouse proteins, especially mAMFR, (an E3 ligase and a cell surface receptor) and mS4 (a component of the 19S "lid" of the proteosome) and mPNGase. Overall the proposed studies should provide much insight into the folding of glycoproteins containing disulfide bonds and the destruction of glycoproteins that cannot achieve their native state. [unreadable] [unreadable]